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Background: This study aimed to evaluate the outcomes of preclinical studies on the safety and immunogenicity of an inactivated COVID-19 vaccine candidate to warrant further clinical evaluation. Method(s): SARS-CoV-2 positive nasopharyngeal swab specimens were confirmed by real-time polymerase chain reaction and next-generation sequencing. The safety and immunogenicity tests of the COVID-19 vaccine were carried out in rats and Rhesus monkeys, and Balb/C mice and Rhesus monkeys, respectively. Result(s): The candidate vaccine was well tolerated and induced promising levels of SARS-CoV-2- specific IgG1, IgG2a, and Granzyme B in Balb/C mice, and anti-SARS-CoV-2 spike IgG and neutralizing antibodies in Rhesus monkeys. Based on cVNT results, the inactivated vaccine in 0.5 and 1 microg/100 microL doses was able to induce a neutralizing effect against the SARS-CoV-2 virus up to a dilution of 1:512 and 1:1000. The protective efficacy of the vaccine candidate was challenged with 2 x108 PFU of live viruses and confirmed by lung CT scan and histopathological evaluations compared to the control group. Repeated intramuscular injection of the candidate vaccine was generally well-tolerated in Rats and Rhesuses. No significant side effects were observed in rats injected with ten full human doses and in the Rhesus monkeys with three full human doses. Conclusion(s): Based on the findings presented in this study, it is recommended that this vaccine be moved into human testing commencing with a phase I clinical trial.Copyright © 2021 Muslim OT et al.
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Intro: Surface and environment disinfection is an important part of infection control strategies, especially in the ongoing COVID-19 pandemic. Ozone, a highly reactive oxidant, is a widely used disinfectant in many industries including food, healthcare and water treatment. It has a broad-spectrum activity and leaves no harmful residues. However, most demonstrated efficacy has been at high ozone levels (>1ppm) which can be harmful to humans in case of exposure. Here, we undertook a study to evaluate if exposure to ozone is effective in inactivating SARS-CoV-2 and feline coronavirus (FCoV) even at low concentrations. Method(s): Ozone at 0.07, 0.1 and 1.2 ppm were evaluated for its virucidal activity against SARS-CoV-2 and FCoV. An ozone gas generator (Medklinn Air + Surface Sterilizer (CerafusionTM Technology), Medklinn, Malaysia) supplied controlled levels of ozone to a custom-built chamber of 1.5 ft3 (1.5ft x 1ft x 1ft) where dry virus films containing 1 x 104 PFU of test virus were exposed to ozone gas for 0.5h, 1h, 3h, 5h, and 8h. The experiment was performed at ambient temperature (23-24oC) and relative humidity (RH) of 55% (FCoV only) and 85% (SARS-CoV-2 and FCoV). Finding(s): At low level of ozone of 0.1ppm, >90% reduction of both viruses was achieved after 3h exposure at 85% and 55% humidity. At 1.2ppm, >90% reduction of both viruses was achieved after 0.5h exposure at 85% humidity. Ozone at 0.07ppm, however, did not show good efficacy as reduction not exceeding 90% was achieved only after 8h exposure at 85% and 55% humidity. Conclusion(s): The study demonstrated that low concentration of ozone of at least 0.1 ppm reduced SARS-CoV-2 and FCoV by >90% when used at 85% humidity. The use of low level ozone presents a safer alternative for disinfecting enclosed spaces and greatly reduces any potential harmful health effects in case of accidental exposure.Copyright © 2023
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Background: The continuous evolution of SARS-CoV-2 in the diverse immune landscape (natural, vaccine, hybrid) is giving rise to novel immune escape mutations. So far, the resulting new variants (BA.1, BA.2, BA.2.12.1) were observed to cause mild infections, however, BA.5 infections are associated with an increased risk of hospitalization.1 Therefore it is essential to investigate the pathogenesis of BA.5. Method(s): Here we compared the pathogenicity of Pre-Omicron (B.1.351) and Omicron (BA.1, BA.2.12.1, and BA.5) variants in wild-type C57BL/6J mice and K18-hACE2 mice. The virus replication kinetics was also studied in human Calu3, pulmonary alveolar type 2 (AT2) cells, and airway organoids (HAO). Cell-to-cell spread of virus was measured by syncytia formation assay and immunohistochemistry (IHC) of infected lungs. Result(s): In the results, infection in C57BL/6J mice showed severe weight loss ( >15%) for B.1.351 infected mice and moderate ( >5%) for BA.5 infected. C57BL/6J mice showed higher virus replication of B.1.351 followed by BA.5, BA.1, and BA.2.12.1. At the peak of virus replication (2 days) plaque-forming units from lung extract of BA.5 infected mice were two, and three logs higher compared to BA.1 and BA.2.12.1 respectively. BA.5 infection was lethal to 80% of infected K18-hACE2 mice, whereas the mice looked normal after infection with BA.1 and BA.2.12.1. BA.5 infected mice showed high virus replication in brain tissue. Surprisingly the syncytia formation assay and IHC for BA.5 was comparable to that of B.1.351, indicating the higher cell-to-cell spread of BA.5 and B.1.351 compared to BA.1 and BA.2.12.1, which is one of the measures of pathogenicity. Calu3 and HAO showed the same trend of virus replication as was observed in-vivo experiments however AT2 cells were found to be resistant to BA.5 replication. Conclusion(s): These results suggest that the BA.5 variant (lineage) of Omicron has the potential to regain the pathogenicity as it shows increased virulence compared to other Omicron sub-variants. Lethal infection of BA.5 in K18-hACE2 mice may be attributed to catastrophic encephalitis and increased cell-to-cell spread.
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Not only since SARS-CoV-2, have transmission routes of viruses been of interest. Noroviruses e.g., can be transmitted via smear infection, are relatively stable in the environment and very resistant to chemical disinfection. Some studies determined the virucidal efficacy of laundering processes, but few studies focused on the virucidal efficacy of dishwashing processes. Here, especially consumer related conditions are of interest. Households for example are a hotspot of norovirus infection and thus a sufficient reduction of these and other viruses from dishes must be insured to avoid an infection via this route. The likelihood of such an event should not be underestimated, since it was shown that the washing machine can be a reservoir for the transmission of extended spectrum beta-lactamase producing bacteria in newborns. Although viruses do not replicate in these devices a transmission via contaminated cutlery e.g., cannot be excluded. Using a consumer related approach to determine the virucidal efficacy of dishwashers, we found a combination of a bleach containing dishwasher detergent, a cleaning temperature of 45 C for 45 min and a rinsing temperature of 50 C, to be sufficient to reduces viral titer of bovine corona virus, murine norovirus and modified vaccinia virus by 4.8, 4.2 and 3.8 logarithmic stages respectively.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
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Background. AZD7442-a combination of 2 human, extended-half-life, SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) (tixagevimab/cilgavimab)-has received US Food and Drug Administration emergency use authorization for COVID-19 prevention in immunocompromised individuals. We evaluated the effect of AZD7442 in prevention and treatment settings in Syrian hamsters challenged with SARS-CoV-2. Methods. Hamsters received intraperitoneal isotype control mAb (2 mg) or AZD7442 (0.002-2 mg) 1 day before intranasal (IN) SARS-CoV-2 challenge (USA-WA1/2020;1x105 plaque-forming units) in prevention;OR control mAb (5 mg) or AZD7442 (0.5-5 mg) 1 day after IN SARS-CoV-2 challenge in treatment. The impact of AZD7442 on lung viral RNA and pathology and AZD7442 serum levels was assessed on Days 3 and 7 post infection. Body weight was recorded daily through Day 7. Results. With AZD7442 prevention, lower lung viral loads were observed compared to controls;at Day 3 post infection, lowest infectious virus titer and viral subgenomic mRNA (sgmRNA) levels were seen with doses >=0.2 mg AZD7442. Concomitantly, increased serum levels of AZD7442 were observed. By Day 7, infectious virus titer and sgmRNA fell below the level of detection (LOD) at all doses tested. Moreover, AZD7442 at doses >=0.2 mg protected hamsters from weight loss versus controls. Lung pathology scores (scale: 0 [normal] to 25 [most severe]) were generally dose dependent, with mean scores of < 2 for AZD7442 versus 10 for controls, indicating less SARS-CoV-2-induced inflammation and alveolar damage in hamsters given AZD7442. Lower AZD7442 doses were associated with mean pathology scores similar to controls. With AZD7442 treatment, infectious virus titers were below the LOD at Day 3 post infection and at Day 7 for sgmRNA, for all doses tested. Mean lung pathology score was <2 for AZD7442 versus 12 for controls. AZD7442 doses >=0.5 mg protected against weight loss relative to controls. Conclusion. In a SARS-CoV-2 challenge model, AZD7442 administered as prevention or treatment led to significantly lower lung viral loads and improved lung pathology, without weight loss. There was also no evidence that AZD7442 mediated antibody-dependent enhancement of disease or infection.
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Purpose: To determine if IgM has a direct effect in preventing SARS-CoV-2 replication in Vero E6 cells, and delaying or preventing disease in infected K18- hACE2 mice. Method(s): 1) Vero E6 cells, grown to confluence in 12 well plates, were used to test the effect of IgM in reducing the number of plaque-forming units (PFU).There were 4 groups: a) 25PFU WA-1 SARS-CoV-2 was combined with 20, 5 or 0.8mug IgM in growth medium, and incubated for 1hr in a final volume of 500ul. 100mul was added to Vero E6 cells in replicate wells and incubated for 1hr;b) 100mul of 20, 5 or 0.8mug IgM was added to Vero E6 cells and incubated. Media was aspirated and the cells were then inoculated with 25PFU WA-1 and incubated for 1hr;c) Virus control - as above, but with no IgM;d) No virus or IgM. FBS growth medium containing Avicel was overlain in the wells and incubated for 48 hours. Virus replication was stopped by incubating with 10% buffered formalin. Following removal of formalin, plates were stained with Giemsa violet, dried, and photographed. 2) A COVID -19 Spike- ACE2 binding assay kit was used to determine if IgM (2ug, 4.5ug, 20ug, 45ug IgM) inhibits the interaction between the Spike-receptor binding domain (S-RBD) and Angiotensin I ConvertingEnzyme 2 (ACE2) receptor. 3) K18-hACE2 mice were divided into 3 groups based on treatment regimen;Group 1: with IgM, No virus;2: with Saline, with virus;3: with IgM, with virus. 35ug IgM was injected intraperitoneal in a single dose, 2 days prior to infection. Mice were innoculated intranasally with 1250 pfu of HK SARS-CoV-2. Result(s): 1) Exposure of 25PFU SARS-CoV-2 to IgM (at all concentrations) prior to incubation with Vero E6 cells, inhibited its replication in Vero E6 cells. When Vero E6 cells were incubated with IgM prior to infection, no plaques were seen in wells with 20ug and 5ug IgM but were observed in wells with 0.8ug IgM. Plaques were also observed in the Virus alone group, but none were seen in the 'No IgM-No virus' group. 2) 45ug IgM/100uls inhibited the binding of S-RBD to ACE2 by ~94-100%, 20ug IgM/100uls inhibited it by ~80%, and 2 or 4.5ug/100ul by ~70-75%. Control without IgM did not inhibit the S-RBD-ACE-2 binding. 3) Pretreatment with a single low dose IgM injection delayed weight loss and mortality. Conclusion(s): IgM inhibits the replication of SARS-CoV-2 in Vero cells in vitro. It also inhibits the interaction between S-RBD that is present on the viral surface and the ACE2 receptor, by binding to S-RBD. A single low dose of IgM given prechallenge delayed disease in infected mice. The discovery that IgM interferes with the formation of the S-RBD-ACE2 complex, and that a single low dose can delay disease, indicates its translational potential as a vaccine/therapeutic to prevent or treat COVID-19.
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Introduction Reprocessing of medical devices becomes increasingly complex, due to higher hygiene requirements. Former studies revealed satisfactory bactericidal properties of UV-C light disinfection in otorhinolaryngological endoscopes. Especially in the context of the current COVID-19 pandemic, virucidal properties are of high importance. In this study the virucidal efficacy of UV-C light disinfection was analyzed using a bacteriophage model. Materials and methods MS-2 bacteriophages were used as surrogate for stable viruses with high tenacity. The bacteriophage samples were irradiated for up to three cycles of 25 seconds by UV-C light (D25, UV-Smart Technologies B.V.). A dilution series of the irradiated test samples was mixed with 1x108 CFU of Escherichia coli. After incubation plaque formation of Escherichia coli were counted and hence the phage concentration was determined. Results The initial contamination of the test samples was 1.8x1012 plaque forming units (pfu). After 25 seconds of UV-C light irradiation, a non-significant Log reduction of 2.2 was found (p = 0,82). After 50 and 75 seconds of UV-exposure a significant Log reduction of 3.4 e.g. 5.1 (p = 0.05 e.g. p = 0.004) was found. The control sample showed a titer of 1.2 x 1010 pfu. Conclusion The tested UV system seems to provide a significant virucidal effect after a short time of exposure. In combination with the results of the bacteriological testing it appears suitable for clinical use as a substitute for current disinfection methods.
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Background and Objectives: SARS-CoV-2 variants of concern (VOC) and interest (VOI) pose a significant threat to public health because the rapid change in the SARS-CoV-2 genome can alter viral phenotypes such as virulence, transmissi-bility and the ability to evade the host response. Hence, SARS-CoV-2 quantification techniques are essential for timely diagnosis and follow-up. Besides, they are vital to understanding viral pathogenesis, antiviral evaluation, and vaccine de-velopment. Materials and Methods: Five isolates of SARS-CoV-2: D614G strain (B.1), three VOC (Alpha, Gamma and Delta), and one VOI (Mu) were used to compare three techniques for viral quantification, plaque assay, median tissue culture infectious dose (TCID) and real-time RT-PCR. 50 Results: Plaque assay showed viral titers between 0.15 ± 0.01×107 and 1.95 ± 0.09×107 PFU/mL while viral titer by TCID 50 assay was between 0.71 ± 0.01×106 to 4.94 ± 0.80×106 TCID /mL for the five SARS-CoV-2 isolates. The PFU/mL titer 50 obtained by plaque and the calculated from TCID assays differed by 0.61 log10, 0.59 log10, 0.59 log10 and 0.96 log10 50 for Alfa, Gamma, Delta, and Mu variants (p≤0.0007), respectively. No differences were observed for the D614G strain. Real-time PCR assay exhibited titers ranging from 0.39 ± 0.001×108 to 3.38 ± 0.04×108 RNA copies/µL for all variants. The relation between PFU/mL and RNA copies/mL was 1:29800 for D614G strain, 1:11700 for Alpha, 1:8930 for Gamma, 1:12500 for Delta, and 1:2950 for Mu. Conclusion: TCID assay was comparable to plaque assay for D614G but not for others SARS-CoV-2 variants. Our data 50 demonstrated a correlation among PFU/mL and E gene RNA copies/µL, units of measure commonly used to quantify the viral load in diagnostic and research fields. The results suggest that the proportion of infectious virions in vitro changes de-pending on the SARS-CoV-2 variant, being Mu, the variant reaching a higher viral titer with fewer viral copies.
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INTRODUCTION AND OBJECTIVE: Neurodegenerative diseases, such as multiple sclerosis (MS), often lead to the development of neurogenic lower urinary tract symptoms (LUTS). We previously characterized neurogenic bladder dysfunction in a mouse model of MS induced by a coronavirus, mouse hepatitis virus (MHV). The objective of this study was to identify genes and pathways linking neuroinflammation in the central nervous system with urinary bladder dysfunction to enhance our understanding of the mechanisms underlying LUTS in demyelinating diseases. METHODS: Adult C57BL/6 male mice (N=12) received either an intracranial injection of MHV (6,000 PFU) or sterile saline (control). The lumbosacral (L6-S2) spinal cord (SC) segments and urinary bladders were collected during acute infection stage (week 1) and at the first peak of demyelination (week 4) after inoculation with the virus. Total RNA was isolated and analyzed using Nanostring nCounter Neuroinflammation panel. The expression levels of 770 genes associated with neuroinflammation were assessed and compared between the specimens. RESULTS: Transcriptome analysis of SC specimens confirmed a significantly increased expression of 132 genes in MHV mice (tens to hundreds fold change) involved in the regulation of astrocyte, microglia and oligodendrocyte functions, neuroinflammation and immune responses. Out of 132 genes up-regulated in the SC, only 2 genes (siglec1, 46-fold in the SC, 2.6-fold at 1 week and 1.8-fold at 4 weeks in the bladder;and zbp1, 568-fold in the SC, 2.8-fold at 1 week and 2.2-fold at 4 weeks in the bladder) were up-regulated in the urinary bladders of MHV-infected mice. Additionally, two genes were significantly up-regulated (ttr, 2.2-fold at 1week and 1.7-fold at 4 weeks;and ms4a4a, 2.3-fold at 1week and 1.6-fold at 4 weeks), and two were down-regulated (asb2, -1.8-fold at 1 week and -1.6-fold at 4 weeks, and myct1, -1.7-fold at 1week and -1.6-fold at 4 weeks) exclusively in the urinary bladders of MHV mice. CONCLUSIONS: Two genes, siglec1 (encodes type 1 transmembrane protein, expressed in microglia and macrophages, promotes neuroinflammation) and zbp1 (encodes a Z-DNA binding protein, plays role in the innate immune response) link the development of neuroinflammation in the central nervous system with neurogenic changes in the urinary bladders of MHV-infected mice. Further research is needed to establish a functional relationship between expression of these genes and neurogenic LUTS.
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Background: A thorough understanding of the inflammatory reaction to SARS-CoV-2 variants, specifically the Delta and Alpha variants, can provide crucial insight into future treatment of individuals infected with these strains. Mice are an effective model for predicting the pathologic processes of these viruses in humans. Design: K18-hACE2 transgenic mice were raised either under normal conditions (control) or infected with either the Alpha (strain B.1.1.7) or Delta (B.1.617.2) variant of SARS-CoV-2, via intranasal challenge with 1000 PFU virus. Mice were then euthanized at days 2 and 6 post-challenge. Lung sections were used for pathological evaluation of H&E stained slides scanned using the Dynamyx software. Blood vessel cross sections were examined and the average number of marginating inflammatory cells per millimeter of vessel were quantified. Additionally, the percentage of total tissue area that was inflamed was calculated. Results: Our data indicates that the Delta variant elicits a strong lymphocytic immune response in the lungs. At two days postchallenge, Inflammation and margination of inflammatory cells could be appreciated in Delta infected mice, but not in Alpha infected mice. The inflammatory infiltrate was composed predominantly of lymphocytes and occasional histiocytes at 2 days. By day 6, marked perivascular inflammation and margination was appreciated, more prominently in Delta (36 lymphocytes per mm. of endothelium) than Alpha infected mice (22 lymphocytes per mm. endothelium) (p=0.022). At this time point, Alpha infected mice showed 4% involvement of pulmonary area by inflammation, compared to 20% for Delta infected mice (p=0.014). Conclusions: This study quantifies the lymphocytic immune response in the lungs to the Alpha and Delta variants of SARS-CoV-2 in mouse models. Both variants showed a lymphocyte predominant inflammatory response. However, the response was much more robust and severe in Delta infected mice compared to Alpha. The results support why the Delta variant is more virulent and fatal compared to Alpha. Ongoing longer term studies and effects in other organs are ongoing and will help to provide further insight into pathogenesis of long COVID-19.
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Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.
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The unprecedented global spread of the severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 is depicting the distressing pandemic consequence on human health, economy as well as ecosystem services. So far novel coronavirus (CoV) outbreaks were associated with SARS-CoV-2 (2019), middle east respiratory syndrome coronavirus (MERS-CoV, 2012), and SARS-CoV-1 (2003) events. CoV relates to the enveloped family of Betacoronavirus (ßCoV) with positive-sense single-stranded RNA (+ssRNA). Knowing well the persistence, transmission, and spread of SARS-CoV-2 through proximity, the faecal-oral route is now emerging as a major environmental concern to community transmission. The replication and persistence of CoV in the gastrointestinal (GI) tract and shedding through stools is indicating a potential transmission route to the environment settings. Despite of the evidence, based on fewer reports on SARS-CoV-2 occurrence and persistence in wastewater/sewage/water, the transmission of the infective virus to the community is yet to be established. In this realm, this communication attempted to review the possible influx route of the enteric enveloped viral transmission in the environmental settings with reference to its occurrence, persistence, detection, and inactivation based on the published literature so far. The possibilities of airborne transmission through enteric virus-laden aerosols, environmental factors that may influence the viral transmission, and disinfection methods (conventional and emerging) as well as the inactivation mechanism with reference to the enveloped virus were reviewed. The need for wastewater epidemiology (WBE) studies for surveillance as well as for early warning signal was elaborated. This communication will provide a basis to understand the SARS-CoV-2 as well as other viruses in the context of the environmental engineering perspective to design effective strategies to counter the enteric virus transmission and also serves as a working paper for researchers, policy makers and regulators.